Kei Kamide

Mutations and common polymorphisms in ADAMTS13 gene responsible for von Willebrand factor-cleaving protease activity

von Willebrand factor (VWF) is synthesized primarily in vascular endothelial cells and secreted into the plasma as unusually large VWF multimers. Normally, these multimers are quickly degraded into smaller forms by a plasma metalloproteinase, VWF-cleaving protease (VWF-CP). Decreases in the activity of this enzyme result in congenital and acquired thrombotic thrombocytopenic purpura (TTP). The human VWF-CP has recently been purified. Cloning of the corresponding cDNA revealed that the 1,427-aa polypeptide is a member of the ADAMTS gene family, termed ADAMTS13. Twelve rare mutations in this gene have been identified in patients with congenital TTP. Here, we report missense and nonsense mutations in two Japanese families with Upshaw–Schulman syndrome, congenital TTP with neonatal onset and frequent relapses. The comparison of individual ADAMTS13 genotypes and plasma VWF-CP activities indicated that the R268P, Q449stop, and C508Y mutations abrogated activity of the enzyme, whereas the P475S mutant retained low but significant activity. The effects of these mutations were further confirmed by expression analysis in HeLa cells. Recombinant VWF-CP containing either the R268P or C508Y mutations was not secreted from cells. In contrast, Q449stop and P475S mutants were normally secreted but demonstrated minimal activity. Genotype analysis of 364 Japanese subjects revealed that P475S is heterozygous in 9.6% of individuals, suggesting that approximately 10% of the Japanese population possesses reduced VWF-CP activity. We report on a single-nucleotide polymorphism associated with alterations in VWF-CP activity; it will be important to assess this single-nucleotide polymorphism as a risk factor for thrombotic disorders.

Genetic variants at the 9p21 locus contribute to atherosclerosis through modulation of ANRIL and CDKN2A/B

UNLABELLED Genome-wide association studies (GWAS) have identified genetic variants contributing to the risk of cardiovascular disease (CVD) at the chromosome 9p21 locus. The CVD-associated region is adjacent to the two cyclin dependent kinase inhibitors (CDKN)2A and 2B and the last exons of the non-coding RNA, ANRIL. It is still not clear which of or how these transcripts are involved in the pathogenesis of atherosclerosis. OBJECTIVE We assessed the hypothesis that 9p21 locus polymorphisms influence the expression of the transcripts in the region (ANRIL, CDKN2A/B) and that these transcripts contribute to atherogenesis through the modulation of proliferation in VSMC. METHODS We genotyped 18 SNPs (r(2)<0.8 and MAF>0.05) across the region of interest: CDKN2A/B and ANRIL, encompassing the CVD-associated region. RNA and DNA were extracted from the blood of 57 volunteers (69-72 years old). Carotid ultrasound was performed in 56 subjects. CDKN2A/B and ANRIL (exons 1-2 and 17-18) expression was measured employing RT-PCR. Gene expression and cell growth were evaluated in cultured VSMC after the siRNA-mediated knock-down of ANRIL. RESULTS The risk alleles for atherosclerosis-related phenotypes were consistently associated with a lower expression of ANRIL when evaluating exons 1-2. Common carotid artery stenosis was associated with a significantly lower (P<0.01) expression of ANRIL (exons 1-2). ANRIL knock-down in VSMC caused significant variation in expression of CDKN2A/B (P<0.05) and reduction of cell growth (P<0.05) in vitro. CONCLUSION Disease-associated SNPs at the 9p21 locus predominantly affect the expression of ANRIL. Overall, our results suggest that several CVD-associated SNPs in the 9p21 locus affect the expression of ANRIL, which, in turn modulate cell growth, possibly via CDKN2A/B regulation.

Impact of High-Normal Blood Pressure on the Risk of Cardiovascular Disease in a Japanese Urban Cohort: The Suita Study

Few prospective studies have examined the association between high-normal blood pressure and cardiovascular disease (CVD) in Asia. We examined the impact of high-normal blood pressure on the incidence of CVD in a general urban population cohort in Japan. We studied 5494 Japanese individuals (ages 30 to 79 years without CVD at baseline) after completing a baseline survey who received follow-up through December 2005. Blood pressure categories were defined on the basis of the ESH-ESC 2007 criteria. In 64 391 person-years of follow-up, we documented the incidence of 346 CVD events. The frequencies of high-normal blood pressure and hypertension Stage 1 and Stage ≥2 were 18.0%, 20.1%, and 10.1% for men and 15.9%, 15.6%, and 8.8% for women, respectively. Antihypertensive drug users were also classified into the baseline blood pressure categories. Compared with the optimal blood pressure group, the multivariable hazard ratios (95% confidence intervals) of CVD for normal and high-normal blood pressure and hypertension Stage 1 and Stage ≥2 were 2.04 (1.19 to 3.48), 2.46 (1.46 to 4.14), 2.62 (1.59 to 4.32), and 3.95 (2.37 to 6.58) in men and 1.12 (0.59 to 2.13), 1.54 (0.85 to 2.78), 1.35 (0.75 to 2.43), and 2.86 (1.60 to 5.12) in women, respectively. The risks of myocardial infarction and stroke for each blood pressure category were similar to those of CVD. Population-attributable fractions of high-normal blood pressure and hypertension for CVD were 12.2% and 35.3% in men and 7.1% and 23.4% in women, respectively. In conclusion, high-normal blood pressure is a risk factor for the incidence of stroke and myocardial infarction in a general urban population of Japanese men.

Genetic variations of regulator of G-protein signaling 2 in hypertensive patients and in the general population.

Objectives Mice deficient in the regulator of G-protein signaling 2 (RGS2) exhibit a strong hypertensive phenotype. We studied whether genetic variations in RGS2 are implicated in hypertension or other phenotypes in Japanese hypertensive individuals and the general population. Methods We sequenced all exons of RGS2 and the promoter region in 953 and 48 hypertensive individuals, respectively. Genotyping by the TaqMan polymerase chain reaction method was performed for six missense or frameshift mutations and common single nucleotide polymorphisms in the general population, with a sample size of 1872 individuals (862 men and 1011 women). Results We identified five novel missense mutations (Q2L; n = 2, Q2R; n = 1, M5V; n = 1, R44H; n = 2, Q78H; n = 1) and one novel frameshift mutation (1925–1926insT; n = 2) in a heterozygous state, in addition to 33 variations including five common single nucleotide polymorphisms. Six missense/frameshift mutations and three common single nucleotide polymorphisms (−638A > G, 1026T > A, 1891–1892delTC) were successfully genotyped in the general population. Mutations Q2L (n = 2), M5V (n = 1), and 1925–1926insT (n = 2) were only identified in hypertensive subjects. Six out of seven individuals with the R44H mutation, which occurs in the amphipathic α-helical domain of RGS2, had hypertension. The results showed a significant association of two common single nucleotide polymorphisms, 1026T > A [TT versus TA + AA: odds ratio (OR) 1.33; 95% confidence interval (CI) 1.02–1.74; P = 0.035] and 1891–1892delTC (I: insertion allele, D: deletion allele, II versus ID + DD: OR 1.47; 95% CI 1.09–1.97; P = 0.012), with hypertension in women by multivariate logistic regression analysis. Conclusion Our results suggest that genetic variations in RGS2 contribute partly to the hypertensive phenotype.

ANRIL: Molecular Mechanisms and Implications in Human Health

ANRIL is a recently discovered long non-coding RNA encoded in the chromosome 9p21 region. This locus is a hotspot for disease-associated polymorphisms, and it has been consistently associated with cardiovascular disease, and more recently with several cancers, diabetes, glaucoma, endometriosis among other conditions. ANRIL has been shown to regulate its neighbor tumor suppressors CDKN2A/B by epigenetic mechanisms and thereby regulate cell proliferation and senescence. However, the clear role of ANRIL in the pathogenesis of these conditions is yet to be understood. Here, we review the recent findings on ANRIL molecular characterization and function, with a particular focus on its implications in human disease.

Uric Acid, Left Ventricular Mass Index, and Risk of Cardiovascular Disease in Essential Hypertension

Elevated serum uric acid (UA) is frequently encountered in individuals with hypertension, but whether the relationship between UA and cardiovascular events is circumstantial or causal remains to be answered. We examined the association between serum UA and left ventricular mass index (LVMI) and investigated prospectively whether the combination of UA and LVMI can predict the incidence of cardiovascular disease (CVD) in asymptomatic subjects with essential hypertension. A total of 619 subjects (mean age, 61 years; 52% female) free of prior CVD were included in this study. A significant association between UA and LVMI was also confirmed in multiple regression analysis (male: F=4.29, P<0.04; female: F=4.24, P<0.05). During follow-up (mean, 34 months), 28 subjects (14 female) developed CVD including myocardial infarction, angina pectoris, congestive heart failure, cerebral infarction, and transient cerebral ischemia. Sex-specific median values were used to separate the higher group from the lower group of UA and LVMI. Kaplan–Meier curves showed a significantly poorer survival rate in the group with higher UA and LVMI (LVMI, male: >126.9, female: >112.0 g/m2; UA, male: >374.7, female: >303.3 &mgr;mol/L; log-rank &khgr;2=13.18; P<0.01). Multivariate Cox regression analysis showed that the combination of higher UA and LVMI was an independent predictor for CVD events (hazard ratio, 2.38; P<0.03). Our findings demonstrate that UA is independently associated with LVMI and suggest that the combination of hyperuricemia combined with left ventricular hypertrophy is an independent and powerful predictor for CVD. The association between UA and CVD events may be introduced in part because of a direct association of UA with LVMI.

Chronic kidney disease as an independent risk factor for new-onset atrial fibrillation in hypertensive patients.

Objective Chronic kidney disease (CKD) has recently been recognized to be a powerful predictor of cardiovascular morbidity and mortality. Atrial fibrillation (AF), which is a common arrhythmia in hypertensives, is associated with increased risks of cardiovascular events and death. However, the association between CKD and the onset of AF has not been fully elucidated. The present study assessed the hypothesis that CKD may influence the onset of AF in hypertensives. Methods A total of 1118 hypertensive patients (mean age, 63 years) without previous paroxysmal AF, heart failure, myocardial infarction, or valvular disease were enrolled. CKD was defined as decreased glomerular filtration rate (<60 ml/min per 1.73 m2) and/or the presence of proteinuria (≥1+). Results During follow-up periods (mean, 4.5 years), 57 cases of new-onset AF were found (1.1% per year). Kaplan–Meier curves revealed that the cumulative AF event-free rate was decreased in the CKD group (log-rank test P < 0.001). By univariate Cox regression analysis, age, smoking, left atrial dimension, left ventricular mass index, and the presence of CKD were significantly associated with the occurrence of AF. Among these possible predictors, CKD (hazard ratio 2.18, P = 0.009) was an independent determinant for the onset of AF in multivariate analysis. Advanced stages of CKD (stages 4 and 5) were strongly related to the increased occurrence of AF. Conclusion The present study demonstrated that the complication of CKD, especially progressed renal dysfunction, was a powerful predictor of new-onset AF in hypertensive patients, independently of left ventricular hypertrophy and left atrial dilatation.

Polymorphism of angiotensin converting enzyme, angiotensinogen, and apolipoprotein E genes in a Japanese population with cerebrovascular disease

The homozygous deletion allele of the angiotensin converting enzyme gene (ACE/DD), homozygous threonine allele of the angiotensinogen gene (AGN/TT), and the epsilon4 allele of the apolipoprotein E gene (apoE/epsilon4) are reported to be associated with ischemic heart disease. Cerebrovascular disease (CVD) is another atherosclerotic disease; and the effects of these polymorphisms on CVD have been confusing. In this study, we investigated whether ACE/DD, AGN/TT, and apoE/epsilon4 genotypes are associated with CVD and whether genetic risk is enhanced by the effect of one upon another. We ascertained these genotypes in patients with cerebral infarction (n = 55) and cerebral hemorrhage (n = 38), diagnosed by brain computed tomography. Control subjects for the infarction group and the hemorrhage group were randomly selected from 583 subjects matched for age, gender, and history of hypertension with patients. Frequency of ACE/DD genotype was higher in the patients with infarction than in the controls (chi2 = 6.1, P < .05). The AGN/TT genotype was not associated with either infarction or hemorrhage, but it increased the relative risk for cerebral infarction in the subjects with ACE/DD genotype (chi2 = 8.0, P < .01, odds ratio; 11.7, 95% confidence intervals: 1.4 to 96.0). There was no significant association between apoE/epsilon4 and CVD. These results suggest that ACE/DD predicts cerebral infarction, but not cerebral hemorrhage, and that AGN/TT enhances the risk for cerebral infarction associated with ACE/DD.

Relationship Between Blood Pressure Category and Incidence of Stroke and Myocardial Infarction in an Urban Japanese Population With and Without Chronic Kidney Disease: The Suita Study

Background and Purpose— Chronic kidney disease (CKD) is increasingly recognized as an independent risk factor for stroke and myocardial infarction (MI). Few studies, however, have examined the relationship between blood pressure (BP) category and these diseases in subjects with and without CKD. Methods— We studied 5494 Japanese individuals (ages 30 to 79, without stroke or MI at baseline) who completed a baseline survey and received follow-up through December 2005. The glomerular filtration rate (GFR) was estimated using the Modification of Diet in Renal Disease study equation modified by the Japanese coefficient. CKD was defined as an estimated GFR <60 mL/min/1.73m2. BP categories were defined by the European Society of Hypertension and European Society of Cardiology 2007 criteria. Results— In 64 395 person-years of follow-up, we documented 346 incidences of cardiovascular diseases (CVD; 213 strokes and 133 MI events). Compared with the GFR (≥90 mL/min/1.73m2) group, the hazard ratios (95% confidential intervals) for stroke were 1.9 (1.3 to 3.0) in the GFR 50 to 59 mL/min/1.73m2 group and 2.2 (1.2 to 4.1) in the GFR <50 mL/min/1.73m2 group. Results for cerebral infarction were similar. Compared with the optimal BP subjects without CKD, the normal BP, high-normal BP, and hypertensive subjects without CKD showed increased risks of CVD and stroke; however the impact of each BP category on CVD (P for interaction: 0.04 in men, 0.49 in women) and stroke (0.03 in men, 0.90 in women) was more evident in men with CKD. Conclusions— CKD may increase the association of BP and CVD in a Japanese urban population.

The renin-angiotensin and adrenergic nervous system in cardiac hypertrophy in fructose-fed rats.

BACKGROUND Hyperinsulinemia and insulin resistance are associated with left ventricular hypertrophy (LVH) and cardiovascular complications in hypertensive subjects. The aim of this study was to explore the mechanisms for LVH including activation of the renin-angiotensin system system (RAS) and the sympathetic nervous system and their activation by insulin using a rat model of hyperinsulinemia and insulin resistance. METHODS Male Sprague-Dawley rats were fed a high-fructose or control diet. The fructose-fed rats (FFR) were divided into four subgroups that were administrated either vehicle or the following antihypertensive drugs (n = 6-8) for 4 weeks: 1) olmesartan, an angiotensin II type 1 (AT1) receptor antagonist; 2) bunazosin, an alpha1-receptor blocker; and 3) hydralazine, a direct vasodilator. RESULTS Fructose feeding induced significant increases in mean systolic blood pressure (BP) levels at 4 weeks (control, 117 v fructose, 131 mm Hg), left ventricular weight, and the sum of the insulin level in response to a glucose tolerance test (2 g/kg). Fructose feeding also increased urinary excretion of epinephrine and norepinephrine, the density of cardiac alpha1-adrenergic receptors, and the content of angiotensin II in the left ventricle. All antihypertensive drugs decreased systolic BP, but only the AT1 receptor antagonist attenuated the development of LVH in FFR. The AT1 receptor antagonist did not affect glucose-mediated insulin responses, but did suppress urinary catecholamine excretion and cardiac alpha1-adrenergic receptor density. CONCLUSIONS Left ventricular hypertrophy in FFR may be less dependent on systemic elevations of BP and more dependent on the RAS and the sympathetic nervous system. Use of an AT1 receptor antagonist might be the most beneficial way to prevent progression of LVH through direct effects on tissue RAS and the sympathetic nervous system in FFR. As these changes occur in a rat model with hyperinsulinemia, insulin may have a role in promoting LVH by activating the local RAS and sympathetic nervous system activity.